The present invention relates generally to the field of molecular biology and genetics. Specifically, the present invention relates to a method for producing recombinant baculovirus expressing a gene of interest by selecting for puromycin resistance that is conferred by that baculovirus.
Baculoviridae comprise a family of viruses that infect insect cells, and can be genetically modified to serve as transfer and expression vectors for a highly productive recombinant protein expression system in the eukaryotic host. (Summers and Smith, Tex. Agric. Exp. Stn. Bull. B1555: 1-55 (1987); Davies, Bio/technology 12:47-50 (1994)). Exemplary baculoviruses include but are not limited to Autographa californica, Trichoplusia ni, Rachiplusia ou, Galleria mellonella and Bombyx mori. Of these, Autographa californica is probably the most fully characterized and widely used. The polyhedrin gene of baculoviruses is expressed at very high levels during the natural viral life cycle, but is dispensable in cell culture. This allows it to be replaced by almost any heterologous nucleotide sequence of interest. Any heterologous nucleotide of interest inserted so as to replace the polyhedrin gene may itself be controlled by the polyhedrin promoter, and thus expressed to similarly high levels.
Advantages of the baculovirus expression system include: (1) avirulence of baculoviruses in humans, (2) facility of protein expression in milligram quantities, and (3) ability of insect cells to grow at ambient temperature without the need for carbon dioxide (Kidd and Emery, 1993). Although both prokaryotic and eukaryotic cells have been used to express foreign genetic material, prokaryotic cells such as Escherichia coli are suitable for expressing foreign genetic material only if the gene product does not require post-translational modification such as glycosylation, phosphorlyation or signal peptide cleavage. Prokaryotic cells do not contain the machinery needed for such post-translational modification. Eukaryotic cells, such as insect cells which baculoviruses infect accomplish most eukaryotic post-translational modifications, including phosphorylation, N- and 0-linked glycosylation, acylation, disulphide cross-linking, oligomeric assembly and subcellular targeting which may be critical for the biological integrity of recombinant proteins (Davies, 1994). In contrast to the more commonly used bacterial expression systems, recombinant baculoviral expression generally produces soluble proteins without the need for induction or specific temperature conditions.
Manipulation of baculovirus genomes at the molecular level is challenging, as they comprise some 130 kb of DNA too large to be amenable to conventional plasmid cloning techniques. The traditional solution to this problem has been to introduce foreign genes by homologous recombination, as a cassette also comprising suitable promoter and termination sequences. This is accomplished by flanking the cassette, in a plasmid vector, by the viral DNA sequences which flank the point at which it is to be inserted. The efficiency of this process is usually less than 1%.
In the past, plaque purification has been used to proceed from this point to a clonal vector. Some traditional procedures are described by Smith et al., U.S. Pat. No. 4, 745,051, Smith et al, U.S. Pat. No. 4,879,236, Summers et al., U.S. Pat. No. 5, 169,784, Guarino et al., U.S. Pat. No. 5,077,214, Kang, U.S. Pat. No. 5,194,376, Matsuura et al, U.S. Pat. No. 5,229,293 and Murphy et al., U.S. Pat. No. 5,516,657, the disclosures of which are herein incorporated by reference. Recently, however, several different techniques have been developed to increase the frequency of recombination into the baculovirus genome. The most successful of these are described, infra.
The baculovirus genome has been reconstituted as a replicon which will propagate in the yeast Saccharomyces cerevisia. This was achieved by inserting a yeast Autonomously Replicating Sequence (ARS) into the polyhedrin locus of the baculovirus genome, along with a CEN (centromeric) sequence, which ensures stable low copy number segregation of the genome by acting as a mitotic centromere, and the URA3 selectable marker, to permit growth in a uracil-free medium. Thus the recombination of a polyhedrin promoter-driven foreign gene may be undertaken in yeast, and the resulting baculovirus genome extracted from the yeast cells and transfected directly into insect cells as a clonal virus. Advantages of this method include near 100% efficiency and the ability to manipulate in a foreign host genes whose products might be toxic to insect cells. However, a large number of manipulations are required making this procedure cumbersome and complicated.
The baculovirus genome has also been reconstituted as a replicon which may propagate in Escherichia coli. In a similar approach to that used in yeast, Luckow et al., J. Virology, 67: 4566-4579, initially demonstrated that the baculovirus genome may be modified to replicate as a large plasmid in Escherichia coli, termed a xe2x80x98bacmidxe2x80x99. This may be performed by recombining a mini-F replicon into the polyhedrin locus, conferring autonomous replication and stable, low-copy number segregation of the genome, and the kanr selectable marker. The target site for the Tn7 bacterial transposon may also be introduced, as an in-frame insertion within the lacZa sequence from a pUC-based plasmid. This bacmid may therefore intra-allelically complement the defective xcex2-galactosidase lacZDM15 of Escherichia coli hosts such as DH 1 OB. However, Escherichia coli DH l OB harboring a bacmid with a foreign gene inserted at this Tn7 site would remain lacZaxe2x88x92, enabling visual selection of colonies containing recombinant bacmids. The overall strategy of such a procedure is to accomplish the recombination and selection steps in the heterologous host, here Escherichia coli, and only then to transfer the finished product to insect cells. This strategy has similar advantages to the yeast system but also involves many steps.
Rather than reconstitute the baculovirus replicon in a heterologous host considered preferable for selection of recombinant species, an in vitro recombination reaction to transfer genes from transfer vectors to the polyhedrin locus of the virus has been developed. Exploiting the Cre recombinase of bacteriophage Pi and its substrate lovp, the gene transfer in this system is achieved by a single enzymatic crossover reaction. Both the target baculovirus genome (vaclox) and the transfer vector are engineered to contain lox sites. These 34 nucleotide sequences direct the Cre enzyme to convert the two substrate DNA molecules into topologically unlinked, recombinant products. The reaction proceeds stoichiometrically, with an efficiency of around 70%. This approach has the great advantage of simplicity, however, the maximum efficiency is only about 70%.
In 1990, Kitts et al., Nucl. Acids Res. 18:5667-5672 (1990) derivatised wild-type baculovirus DNA, which exists as a covalently closed circular double-stranded molecule, by introducing a unique restriction site (Bsu361) at the polyhedrin locus. Linearising the baculovirus genome using this restriction site reduces the infectivity of the viral DNA on transfection into insect cells, however, cotransfection with a transfer vector driving recombination into the polyhedrin locus produces a three fold higher proportion of recombinant viruses.
In this strategy, the baculovirus containing the Bsu361 site was designated ACRP-SC (for single cut), and the two double crossover events transferring the foreign gene to be expressed, along with its own copy of the polyhedrin promoter and terminator sequences, are also present. About 10 to 25% of the progeny viruses from such a cotransfection are recombinant, but it should be noted that this is due to the reduced background of wild type viruses rather than an increase in the absolute number of recombinants.
A subsequent development of this system effectively combines it with both a visual (lacZ-based) and a replication-based selection strategy. Kitts et al., Biotechniques 14:810-817 (1993). Recombination occurs between a conventional transfer vector and a derivative of the wild type genome, containing lacZ at the polyhedrin locus and two further Bsu361 sites in the polyhedrin flanking sequences (designated BacPAK6). Bsu361 digestion of this baculovirus DNA not only linearizes the molecule, but also removes two genomic fragments, thus disrupting the open reading frame ORF-1629. This gene is essential for baculovirus replication, so, with the two Bsu361 fragments removed from the genome, competent viruses will only be reconstituted by recombination with the transfer vector, whereby an intact ORF-1629 will be restored to the genome. Furthermore, recombinant viruses will form white plaques on a background of blue plaques from BacPAK6 DNA.
This strategy yields recombinant viruses at a frequency of 85-99%. The majority of the background may be attributed to contamination with undigested BacPAK6 DNA, which will be substantially more infectious than linear forms of the genome. Viruses which offer the prospect of utility for future work, which are likely to be a small minority of the total number of viruses generated, may then be purified by conventional technology (plaque assays). Therefore this method offers a good combination of simplicity and efficiency, and will be our technique of choice for converting arrayed cDNAs into expression vectors.
One particular problem associated with baculovirus expression systems is that they may produce apoptosis or cell lysis of infected cells. A baculovirus apoptosis resistance gene (p35 gene) has been identified that confers increased viral yield in certain cell lines. Recombinant baculoviruses bearing this gene can selectively amplified (up to 106 fold) in appropriate hosts.
Despite the useful combination of high yields and authenticity of processing associated with baculovirus expression systems, purification of the desired product from baculovirus infected cells is no easier than from any other eukaryotic system. Many expression vectors have been developed enabling synthesis of the antigen of interest as a fusion with a polypeptide facilitating purification. For instance, protein A fusion proteins may be affinity purified on IgG, polyarginine fusion proteins may be purified by cation exchange, polyhistidine fusions may be purified by virtue of their chelation of zinc ions, xcex2-galactosidase fusions, and other fusions to specifically immunogenic partners, may be purified by immunoaffinity, and xcex2-galactosidase, maltose binding protein and glutathione-S-transferase (GST) fusion proteins may be purified by substrate affinity. Other epitope tags such as those recognized by the EE or Glu-Glu antipeptide antibody may also be used. This antibody was raised against a peptide that comprises the major tyrosine phosphorylation site of polyoma middle t antigen. This tag has been used extensively in many recombinant applications. Some investigators report that over 90% of attempts to purify EE-tagged proteins have been successful, most of these to over 50% purity with no other chromatographic steps (Jim Litts and Robin Clark unpublished data).
The epitope for this tag has been fully characterized and optimized by NEmotope peptide scan analysis (Mario Geysen, John Wang and Robin Clark unpublished data). The antibody has a moderate affinity for the EE tag (Kd-2xc3x9710xe2x88x927) hich allows rapid elution of tagged proteins by free peptide under non-denaturing conditions while retaining efficient binding of proteins in crude lysates. While sually placed at the N-terminus, the EE tag is also recognized when placed at the C-terminus or at internal portions of the protein. An antibody to the tag is also useful or immunoprecipitates, inumunoflourescence and western blots. Moreover, the ntibody is produced at high levels by the EE hybridoma. Some have reported that 10 preps routinely yield 3-5 g of purified antibody. The EE tag has the additional advantage of containing a strong tyrosine phosphorylation substrate for protein kinases such as src. Such a tag may be directly labeled with radioactive phosphate or detected with an anti-phosphotyrosine antibody.
Several resistance genes have been used as selectable markers in a variety of expression vectors to enhance selection of recombinants. (Old and Primrose, Principles of Gene Manipulation, Oxford: Blackwell Scientific Publications, 1991, pp. 259-262; Karreman, Gene 218 (1-2): 57-61 (1998)). In 1993, a novel puromycin-resistancedeterminant (pur8) was isolated from the puromycin gene cluster that encodes the puromycin biosynthetic pathway from Streptomyces alboniger and expressed in Streptomyces lividans. The gene induced antibiotic resistance that was highly specific for puromycin. The polypeptide product referred to as Pur8 consists of 14 possble transmembranexe2x80x94spanning segments, and shows significant similarities to other known or putative transmembrane proteins, including several which confer drug resistance in a variety of antibioticxe2x80x94producing Streptomyces, Gram-positive and Gram-negative bacteria, and some solute transporters of prokaryotic and eukaryotic origin. (Tercero, et al., Eur. J Biochem. 218(3): 963-971 (1993)) The novel puromycin acetyltransferase, has been used as a selectable marker gene, and can be expressed under the control of the Drosophila melanogaster hsp 70 promotor or AcMNPV ie-l promoter which is active in Spodopterafrugiperda cells in the absence of virus infection. Expression vectors have been constructed which co-express two genes from separate promoters, the pac gene which confers resistance to puromycin and a selectable baculovirus gene which inhibits apoptosis. (McLachlin and Miller, In Vitro Cell Dev. Biol. Anim. 33(7):575-579 (1997)) The puromycin resistance gene (PAC) has also been used as a positive selectable marker in mammalian cells, (Karreman, Gene 218(1-2): 57-61 (1998)) and in single-transcript vectors, where it is expressed from the internal ribosomal entry site of encephalomyocarditis virus (Levenson, et al., Hum. Gene Ther., 9(8): 1233-1236 (1998)).
This invention is a method for producing recombinant baculoviruses, without utilizing cloning steps, expressing genes of interest by inserting a selectable marker gene, specifically the puromycin resistance (pac) gene, adjacent to the gene of interest into the baculovirus genome, and selecting for viruses that express the selectable marker. The majority of the selected viruses should express the gene of interest. Selection of the recombinant virus in the presence of a selection condition, such as puromycin-containing cell culture media, simplifies virus production by eliminating the need for cloning and testing the virus for gene expression.
In one aspect, the present invention features a novel baculovirus expression system especially adept at expressing heterologous genetic material in a host. Generally, such systems comprise one or more selectable marker genes, a promoter such as a polyhedrin promoter of a polyhedrin structural gene or a fragment thereof, a transcription terrnination sequence such as a transcription termination sequence of a polyhedrin structural gene or fragment thereof, a cloning site operable to accept foreign genetic material, optionally a tag and optionally one or more restriction sites. Exemplary selectable marker genes include the puromycin resistance (pac) gene, and exemplary tags include the EE tag sequence. In preferred embodiments, the ORF-1629 gene is also present in the baculovirus expression system and is used as a second selectable marker gene. The invention features a method for producing recombinant baculovirus in which a DNA fragment containing foreign genetic material is used to combine preferably two selectable marker genes into a single DNA fragment, which when co-transfected with Baculovirus DNAs into insect host cells, yields a recombinant baculovirus which can be isolated by selection of selectable marker genes. Therefore, the present invention can be used to produce recombinant baculovirus without utilizing cloning steps, and thereby is useful for automated high throughput expression systems.
In a second aspect, the present invention features a method for producing a recombinant baculovirus expression system comprising the steps of (a) providing a baculovirus transfer vector comprising a puromycin resistance (pac) gene, a promoter such as a polyhedrin promoter of a polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as a transcription termination sequence of a polyhedrin structural gene or fragment thereof, a cloning site operable to accept foreign genetic material, optionally a tag and optionally one or more restriction sites, and (b) ligating foreign genetic material into the baculovirus transfer vector at the cloning site such that the foreign genetic material is under the control of the promoter.
In a third aspect, the present invention features a method for expressing foreign genetic material comprising the steps of: (a) providing a baculovirus transfer vector comprising a puromycin resistance (pac) gene, a promoter such as a polyhedrin promoter of a polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as a transcription termination sequence of a polyhedrin structural gene or fragment thereof, a cloning site operable to accept foreign genetic material, optionally a tag and optionally one or more restriction sites, (b) ligating foreign genetic material into the baculovirus transfer vector at the cloning site such that the foreign genetic material is under the control of the promoter, and (c) transfectipg the baculovirus transfer vector comprising the foreign genetic material into a host cell. In preferred embodiments, the present invention is adapted to automation and thereby is useful for high throughput expression.
In a fourth aspect, the present invention features a method for determining the function of foreign genetic material comprising the steps of: (a) providing a baculovirus transfer vector comprising a puromycin resistance (pac) gene, a promoter such as a polyhedrin promoter of a polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as a transcription termination sequence of a polyhedrin structural gene or fragment thereof, a cloning site operable to accept foreign genetic material, optionally a tag and optionally one or more restriction sites, (b) ligating foreign genetic material into the baculovirus transfer vector at the cloning site such that the foreign genetic material is under the control of the promoter, (c) transfecting the baculovirus transfer vector comprising the foreign genetic material into a host cell, and (d) observing the biological results produced thereby. In preferred embodiments, the present invention is adapted to automation and thereby is useful for high throughput analysis.
In a fifth aspect, the present invention features genes and fragments thereof, nucleotide sequences, and gene products obtained by way of the method of the present invention. The present invention features expressing selected nucleotide sequences in a host organism. Those of skill in the art will readily appreciate that the gene products of such nucleotide sequences may be isolated and purified using techniques known to those skilled in the art. Such isolation and purification techniques may be optimized according to some of the methods described herein. Such gene products may exhibit biological activity as pharmaceuticals and other similar functions.